Compositions and methods for stem cell chondrogenesis

Abstract

Among the various aspects of the present disclosure is the provision of compositions and methods for stem cell chondrogenesis. An aspect of the present disclosure provides for a method of differentiating pluripotent stem cells (e.g., induced pluripotent stem cells (iPSCs), human induced pluripotent stem cells (hiPSCs)) into chondrocyte-like cells (e.g., cartilage).

1 . A method of generating a population of chondrocyte or chondrocyte-like cells, the method comprising:
 (i) culturing a population of pluripotent stem cells in a mesoderm differentiation medium to produce a population of chondroprogentior cells; and   (ii) culturing the population of cells obtained from step (i) in a chondrogenic medium comprising a transforming growth factor beta, a WNT signaling inhibitor and optionally bone morphogenic protein and optionally an inhibitor of the microphthalmia-associated transcription factor (MITF) pathway to produce a population of chondrocyte or chondrocyte-like cells.   
     
     
         2 . The method of  claim 1 , wherein the pluripotent stem cells are induced pluripotent stem cells (iPS). 
     
     
         3 . The method of  claim 1 , wherein the pluripotent stem cells are embryonic stem cells. 
     
     
         4 . The method of  claim 1 , wherein the population of chondroprogenitor cells in step (ii) is prepared in a 3D culture. 
     
     
         5 . The method of  claim 1 , wherein the transforming growth factor beta is TGF-β3. 
     
     
         6 . The method of  claim 1 , wherein the WNT signaling inhibitor is WNT-C59. 
     
     
         7 . The method of  claim 1 , wherein the MITF inhibitor is ML329. 
     
     
         8 . The method of  claim 1 , wherein the bone morphogenic protein is BMP-4. 
     
     
         9 . (canceled) 
     
     
         10 . The method of  claim 1 , wherein the method does not require prospective sorting or expansion of the isolated population of chondrocyte or chondrocyte-like cells and wherein off-target cells and/or chondrocyte hypertrophy is reduced in the population of chondrocyte or chondrocyte-like cells relative to a population of chondrocyte or chondrocyte-like cells produced by a method which does not inhibit WNT signaling and/or MITF signaling in step (ii). 
     
     
         11 . The method of  claim 1 , wherein the base chondrogenic medium media is a mix of DMEM and F12. 
     
     
         12 . The method of  claim 1 , wherein TGF-β3 is present at a concentration of about 10 ng/ml. 
     
     
         13 . The method of  claim 1 , wherein the WNT-C59 is present at a concentration of about 1 μM. 
     
     
         14 . The method of  claim 1 , wherein the ML329 is present at a concentration of about 1 μM. 
     
     
         15 . The method of  claim 1 , wherein the BMP4 is present at a concentration of about 20 ng/ml. 
     
     
         16 . The method of  claim 1 , wherein the chondroprogenitor cells are cultured in the chondrogenic differentiation medium for about 1 to about 56 days. 
     
     
         17 . The method of  claim 1 , wherein the PS cells are genetically modified. 
     
     
         18 . A population of chondrocyte or chondrocyte-like cells, which is produced by a method of  claim 1 . 
     
     
         19 . A method of treating a subject in need thereof, comprising administration of chondrocyte or chondrocyte-like cells produced by the method according to  claim 1 , wherein the subject has a cartilage-related disease, disorder, or condition. 
     
     
         20 . The method of  claim 19 , wherein the cartilage-related disease, disorder or condition is arthritis, osteoarthritis, rheumatoid arthritis, joint injuries, cartilage defects, or growth-related abnormalities or dysplasias. 
     
     
         21 .- 26 . (canceled) 
     
     
         27 . A cell pellet or matrix comprising chondrocyte or chondrocyte-like cells produced according to  claim 1 , wherein the pellet or matrix comprising the cells has a reduced population off-target cells and have increased heterogeneity compared to chondrocyte-like cells not treated with the Wnt inhibitor or MITF inhibitor.